MM induces STING-mediated activation of macrophages via mtDAMPs. (A) Schematic of experimental design. Murine 5TGM1 cells (1 × 10⁶) were injected into KaLwRij mice. (B) At 28 days, BM samples were taken and analyzed for 5TGM1 engraftment by GFP⁺ cells. (C) BM macrophages were FACS-purified (GR1⁻, F4/80⁺, CD115^int), and RNA was extracted for analysis by quantitative real-time PCR (qRT-PCR). (D) Relative gene expression of GBP2, IFIT3, and IRF7 in FACS-purified macrophages. (E) Large and small EV were removed from 5TGM1-CM by centrifugation and filtering through a 100 kDa Amicon Ultra-15 Centrifugal Filter. (F) BMDM were then treated with filtered CM for 6 hours followed by RNA extraction for analysis using qRT-PCR of GBP2, IFIT3, and IRF7. Relative gene expression of GBP2, IFIT3, and IRF7 in BMDMs treated with mtDAMPs (10 μg) (G) or CpG ODN 1826 (n = 4) (H). Data indicate mean ± SD. Statistics are presented as Mann-Whitney U test. (I) BMDM were pretreated with STING inhibitor H-151 (10 μM) for 2 hours before treatment with either CpG ODN 1826 or mtDAMPs (10 μg) for 6 hours. RNA was extracted and gene expression was analyzed via qRT-PCR. Relative gene expression of GBP2, IFIT3, and IRF7 in control untreated BMDMs, CpG (n = 4). Statistics are presented as 2-way analysis of variance with Šidák post hoc multiple comparisons test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. FITC, fluorescein isothiocyanate; FSC, forward scatter; SSC, side scatter.